| ObjectHuang,D. Y. etc purified and identified a novel NADP - dependent retinol dehydrogenase/reductase(NRDR) from rabbit liver cytosol in 1997. NRDR has much higher substrate specificity and affinity than reported alcohol dehydrogenase ( ADH ) and short - chain alcohol de-hydrogenase/reductase (SCAD) , and its activity is widely spread in mammal liver, but rabbit is the most. The full - length NRDR cDNA of rabbit and mouse is approximate about 1200bp,and the full - length NRDR cDNA of human is about ISOObp. The size of their coding region is identical (783bp) ,and encoding a protein of 260 amino acids. They had been cloned and submitted to GenBank ([ AB045133 ] [ BC003484] [ AB045131 ] ) . Here we report the whole process of the cDNA cloning and the analysis of its gene structure.MethodsTotal RNA isolation, purity identification. Total RNA was isolated from human and mouse liver, using a total RNA purification kit ( RNeasy Mini Kit) , and quantitated on the basis of the absorbance at 260/280 nm . The 260/280 value of human liver total RNA is 1. 927 and that of mouse is 1.920. The concentration of the total RNA is respectively 0.768ug/ul and 0.902ug/ul.The amplification of NRDR cDNA fragment. The degenerate primers for PCR were designed from consentient nucleic acid sequences of human and mouse NRDR coding region. One 630bp DNAfragment of mouse and two different fragments (630bp and 400bp )of human were gel - purified and cloned into pGEM - T vector ,then se-quenced by TaKaRa. The fragment of mouse is 635bp, and the two fragments of human are 635bp and 377bp. Further analysis indicated that the 377bp fragment was lack of successive 258bp comparing with 635bp.Amplification of 3' , 5' - end of the human NRDR and NRDRiso cDNA.3' - RACE. 3 ' - RACE reactions were performed using the TaKaRa 3' - full RACE core set following the manufacturers instruction. First, the template for 3' - RACE reaction was human liver cD-NA prepared using TaKaRa AMV Reverse Transcriptase and Oligo dT -3sites Adaptor primer. Then,with the common 291bp part of 377bp and 635bp fragment as template designed a pair of primers. Two 900bp and 650bp of PCR products were obtained and both fragments were subcloned into pGEM ?T plasmid for sequencing.5' - RACE. 5 ' - RACE reactions were performed using the TaKaRa 5' - full RACE core set following the manufacturer^ instruction. First, with the lacking 258bp part of the 635bp fragment as template , we designed one 5 ' - end - phosphorylated primer . Synthesized 1 st strand cDNA by reverse transcription from target mRNA u-sing 5 ' - end - phosphorylated RT Primer which is specific to the target RNA. Second, performed the degradation of hybrid DNA - RNA to separate RNA by treatment with RNase H. Third, carried out the circularization of single - stranded cDNA or formation of concatemers by RNA Ligase. Four, nested 5' - RACE reactions were performed according to the manufacturers instructions; first with primers Fl and Rl, and then a nested RACE reaction with primers F2 and R2. TheRACE products were subcloned into pGEM - T vectors and se-quenced. Meanwhile, with the border of lacking part as template, we designed another 5 ' - end - phosphorylated primer and two pairs of nested primers. The nested 5 ' - RACE reactions were performed with the conditions described immediately above, and obtained a 300bp fragment. PCR product was cloned into pGEM - T vector and the insert was sequenced.Genomic organization, sequence analysis and chromosomal localization. DNA sequence analysis was performed using online NCBI blast nucleotide and local software Jellyfish. The sequence homology search was conducted at the protein level using the BLAST network service of the National Center for Biotechnology Information (NCBI). The sequence alignment among proteins was performed using the program Mac Vector ( OXOL MOL COM). Protein subsequence motifs were identified using the network service SMART (URL: http:// coot, embl - heidelberg. de/SMART/). Prosite information for NRDRiso was analyzed using t... |
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